Otitis media (OM) is the most common childhood bacterial infection and also the leading cause of conductive hearing loss in children. Inflammation in the middle ear caused by microorganisms is a hallmark of OM. Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen that causes OM. However, the pathogenesis of OM induced by NTHi is not fully understood. Our long-term objective is to elucidate the molecular mechanisms by which inflammation is induced and regulated in the pathogenesis of OM so that novel therapeutic strategies can be further developed for treating OM. Our previous studies showed that Toll- like receptor (TLR2) plays a key role in mediating NTHi-induced inflammatory response; however, blocking TLR2 signaling may result in some unwanted detrimental side effects because appropriate immune response mediated by TLR signaling is also required for host defense against bacterial pathogens. For instance, obvious abnormal macrophage physiology, abnormal cytokine secretion and decreased susceptibility to bacterial infection were observed in TLR2 deficient mice. Thus, identifying a non-TLR2-mediated pathway as an ideal therapeutic target for OM is urgently needed. Interestingly, our recent preliminary data showed that epidermal growth factor receptor (EGFR) is also required for NTHi-induced NF-?B activation and inflammatory response via PI3K/AKt and MKK3/6-p38 pathways in human middle ear epithelial cells (HMEEC) in vitro and mouse middle ear in vivo. These encouraging preliminary results have thus laid a solid foundation for us to fully investigate the regulatory mechanisms by which NTHi-induced NF-??B-dependent inflammation is mediated by EGFR via two distinct signaling pathways including PI3K/Akt and MKK3/6-p38 in the pathogenesis of OM in vitro and in vivo (short-term objective and hypothesis). Aim 1. Determine the requirement of EGFR in mediating NTHi-induced activation of NF-??B and inflammatory response in vitro and in vivo. Aim 2. Determine the requirement of PI3K/Akt in mediating EGFR-mediated activation of NF-??B and inflammatory response by NTHi in vitro and in vivo. Aim 3. Determine the requirement of MKK3/6-p38 pathway in mediating EGFR- mediated activation of NF-??B and inflammatory response by NTHi in vitro and in vivo. Given that EGFR inhibitors (such as Gefitinib and erlotinib) have been approved in US for treatment of lung cancer, the proposed studies will not only provide novel insights into the molecular mechanisms underlying the regulation of inflammation in OM, but also lead to development of novel therapeutic strategy for the treatment of OM. Relevance: Nontypeable Haemophilus influenzae (NTHi) is an important human bacterial pathogen that causes Otitis media (OM), the most common childhood infection and the leading cause of conductive hearing loss, which causes a substantial burden on community health programs and links to lower scores in cognitive ability, linguistic skills and school performance. Despite aggressive treatment with antimicrobial agents, approximately 10% of acute OM progress to chronic OM with effusion and inappropriate antibiotic treatment contributes to the worldwide emergence of antibiotic-resistant strains of NTHi. In addition, development of a vaccine for preventing NTHi- induced OM still remains a great challenge. Therefore, development of novel therapeutic strategies for the treatment of OM is urgently needed. The proposed studies will not only bring new insights into our understanding of how inflammation is tightly controlled in OM but may also lead to developing novel therapeutic strategies for treatment of OM.